肺炎衣原体的分离、传代及应用

发布时间：2018-10-18 14:47

【摘要】： 第一部分:PBMC中肺炎衣原体的分离、培养及传代目的:从外周血单个核细胞中分离肺炎衣原体,进行培养、传代、扩增。方法:分离PBMC,用37%PEG使肺炎衣原体阳性的PBMC裂解释放出肺炎衣原体,然后与Hep-2细胞一起离心,继续培养Hep-2细胞。再将Hep-2细胞冻融破碎后,放入到新的Hep-2细胞培养瓶中进行离心,以完成肺炎衣原体的一次传代,然后以同样的方法进行二次传代。用MIF及PCR法检测Hep-2细胞中的肺炎衣原体抗原。结果:MIF法检测肺炎衣原体感染后的Hep-2细胞,其胞内肺炎衣原体抗原阳性MIF法检测一次传代及二次传代的Hep-2细胞,其胞内肺炎衣原体抗原均阳性;PCR法检测肺炎衣原体DNA阳性。结论:该方法可以实现PBMC中肺炎衣原体的分离,并实现进一步的培养及传代。第二部分自制肺炎衣原体抗原的应用评估目的:对自制Cpn-Ag检测血清抗体进行评估。方法:粗制Cpn-Ag,Dot-ELISA法观察自制Cpn-Ag与进口单抗的结合反应。用自制Cpn-Ag和进口Cpn-AR39-Ag通过微量免疫荧光法同时检测血清标本268例(Cpn-IgM、IgA、IgG)。结果:Dot-ELISA法结果显示自制Cpn-Ag能与进口单抗发生结合反应。自制Cpn-Ag与进口Cpn-AR39-Ag检测血清中Cpn-IgA、IgG、IgM抗体滴度(见表2、4、6),两抗原检测一致率均达90%以上,以进口AR39-Ag检测为标准,自制抗原检测灵敏度、特异度均在90%以上。阳性似然比均大于10,在IgM阳性似然比高达30,阴性似然比均小于0.1。采用配对卡方检验,显示P值均＞0.05,表明自制与进口Cpn-Ag检测血清IgA、IgG、IgM结果没有差别。同时采用一致性检验,Kappa值＞0.75,认为两者具有高度一致性。两抗原检测阳性结果抗体滴度总体水平无差异。结论:自制Cpn-Ag检测血清Cpn-IgA、IgG、IgM具有较高的灵敏度和特异度,与进口AR-39-Ag检测具有高度一致性,自制Cpn-Ag可能可以替代进口Cpn-AR39-Ag用于血清样本的检测,以诊断临床Cpn感染。
[Abstract]:Part I: isolation, culture and passage of chlamydia pneumoniae from PBMC objective: to isolate, culture and amplify chlamydia pneumoniae from peripheral blood mononuclear cells (PBMC). Methods: chlamydia pneumoniae was released from chlamydia pneumoniae positive PBMC by 37%PEG by PBMC, then centrifuged with Hep-2 cells to culture Hep-2 cells. After freezing and thawing Hep-2 cells were put into a new Hep-2 cell culture bottle for centrifugation to complete the first passage of Chlamydia pneumoniae and then to carry on the second passage in the same way. Chlamydia pneumoniae antigen in Hep-2 cells was detected by MIF and PCR. Results: the Hep-2 cells infected with Chlamydia pneumoniae were detected by MIF, and the primary and second passages of Hep-2 cells were detected by MIF method, and chlamydia pneumoniae DNA was detected by PCR method. Conclusion: this method can be used to isolate chlamydia pneumoniae from PBMC and further culture and subculture. The second part is the application evaluation of chlamydia pneumoniae antigen. Objective: to evaluate the detection of serum antibodies by self-made Cpn-Ag. Methods: the binding reaction of self-made Cpn-Ag with imported McAb was observed by crude Cpn-Ag,Dot-ELISA method. A total of 268 serum samples (Cpn-IgM,IgA,IgG) were simultaneously detected by self-made Cpn-Ag and imported Cpn-AR39-Ag by micro-immunofluorescence assay. Results: the results of Dot-ELISA showed that self-made Cpn-Ag could react with imported McAbs. The titer of Cpn-IgA,IgG,IgM antibody in serum was detected by self-made Cpn-Ag and imported Cpn-AR39-Ag (see Table 2 / 4). The detection consistency rate of the two antigens was more than 90%. The sensitivity and specificity of self-made antigen detection were above 90% according to the standard of imported AR39-Ag detection. The positive likelihood ratio was more than 10, the positive likelihood ratio was 30 in IgM, and the negative likelihood ratio was less than 0.1. The results of paired chi-square test showed that P values were all more than 0.05, which indicated that there was no difference between self-made and imported Cpn-Ag in the detection of serum IgA,IgG,IgM. At the same time, the consistency test, Kappa value > 0. 75, think they have a high consistency. There was no difference in the titer of antibody between the two antigens. Conclusion: the detection of serum Cpn-IgA,IgG,IgM by self-made Cpn-Ag has high sensitivity and specificity, which is consistent with the detection of imported AR-39-Ag. Self-made Cpn-Ag may replace imported Cpn-AR39-Ag for detection of serum samples in order to diagnose clinical Cpn infection.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R374

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